Isolation of lactoferrin from milk

ABSTRACT

PCT No. PCT/EP95/00583 Sec. 371 Date Oct. 16, 1996 Sec. 102(e) Date Oct. 16, 1996 PCT Filed Feb. 16, 1995 PCT Pub. No. WO95/22258 PCT Pub. Date Aug. 24, 1995The invention provides methods for purification of human lactoferrin from milk, especially milk of nonhuman species, and for separation of human lactoferrin from undesired macromolecular species present in the milk, including separation from nonhuman lactoferrin species.

FIELD OF THE INVENTION

The invention relates to the purification of lactoferrin from milk,particularly the purification of human lactoferrin from the milk oftransgenic non-human animals expressing a human lactoferrin polypeptideencoded by a transgene.

BACKGROUND

Recent advances in the field of molecular biology allow the productionof transgenic animals (i.e., non-human animals containing an exogenousDNA sequence in the genome of germline and somatic cells introduced byway of human intervention). Differences in the regulation of theseforeign genes in different cell types make it possible to promote thedifferential expression of the foreign gene in a preselected tissue,such as the mammary gland, for ease of isolation of the protein encodedby the foreign gene, for a desired activity of the foreign gene productin the selected tissues, or for other reasons.

An advantage of transgenic animals and differential gene expression isthe isolation of important proteins in large amounts, especially byeconomical purification methods. Such proteins are typically exogenousto the transgenic animal and may comprise pharmaceuticals, foodadditives, nutritional supplements, and the like. However, exogenousproteins are preferably expressed in tissues analogous to those in whichthey are naturally expressed. For example, exogenous milk proteins(e.g., lactoferrin) are preferably expressed in milk-forming tissues inthe transgenic animal. As a result, difficult isolation problems arepresented because the exogenous protein is often expressed in the tissueor bodily fluid containing an endogenous counterpart protein (if itexists), and possibly other undesired contaminant species which may havevery similar physicochemical properties. Moreover, many exogenousproteins must be substantially purified from other species, frequentlypurified to homogeneity, prior to their use as pharmaceuticals or foodadditives.

For example, the production of transgenic bovine species containing atransgene encoding a human lactoferrin polypeptide targeted forexpression in mammary secreting cells is described in WO91/08216,published Jun. 13, 1991. The purification of human lactoferrin (hLF)from a transgenic animal containing a functional endogenous bovinelactoferrin (bLF) gene and a transgene encoding the expression of hLF iscomplicated by the presence of endogenous bLF which has physicochemicalproperties similar to human lactoferrin. Even in a transgenic bovinelacking a functional endogenous bLF gene (e.g., as a result ofhomologous gene targeting to functionally disrupt the endogenous bLFalleles), it is frequently desirable and/or necessary to purifytransgene-encoded hLF from other biological macromolecules andcontaminant species. Since hLF has potential pharmaceutical uses and maybe incorporated in human food products as a nutritive supplement, useswhich typically require highly purified hLF, it is imperative thatmethods be developed to purify hLF from milk, especially from milk ormilk fractions of transgenic nonhuman animals such as bovine species.

Human lactoferrin is a single-chain glycoprotein which binds ferricions. Secreted by exocrine glands (Mason et al. (1978) J. Clin. Path.31: 316; Tennovuo et al. (1986) Infect. Immunol. 51: 49) and containedin granules of neutrophilic leukocytes (Mason et al. (1969) J. EXP. Med.130: 643), this protein functions as part of a host nonspecific defensesystem by inhibiting the growth of a diverse spectrum of bacteria. hLFexhibits a bacteriostatic effect by chelation of the available iron inthe medium, making this essential metal inaccessible to themicroorganisms (Bullen et al. (1972) Brit. Med. J. 1: 69; Griffiths etal. (1977) Infect. Immunol. 15: 396; Spik et al. (1978) Immunology 8:663; Stewart et al. (1984) Int. J. Biochem. 16: 1043). Thebacteriostatic effect may be blocked if ferric ions are present inexcess of those needed to saturate the hLF binding sites.

Lactoferrin is a major protein in human milk (present at a concentrationof about 1.5 mg/ml) and may play a role in the absorption of dietaryiron by the small intestine. Essentially all of the iron present inhuman breast milk is reported to be bound to hLF and is taken up at veryhigh efficiency in the intestine as compared to free iron in infantformula (Hide et al. (1981) Arch. Dis. Child. 56: 172). It has beenpostulated that the efficient uptake of hLF-bound iron in humans is dueto a receptor in the duodenum (Cox et al. (1979) Biochim. Biophys. Acta588: 120). Specific lactoferrin receptors have been reported on mucosalcells of the small intestine of human fetuses (Kawakami and Lonnerdal(1991) Am. J. Physiol. 261: G841.

hLF from human colostrum is available commercially (Calbiochem, LaJolla, Calif. and other vendors) as a lyophilisate for researchapplications in small amounts ( mg and 25 mg vials). The amino acidsequence of hLF has been reported (Metz-Boutigue et al. (1984) Eur. J.Biochem. 1451: 659), and WO91/08216 reports an hLF sequence having somesequence inconsistencies with the previous report of Metz-Boutigue etal. hLF comprises two domains, each comprising an iron-binding site andan N-linked glycosylation site. These domains show homology with eachother, consistent with an ancestral gene duplication and fusion event.hLF also shares extensive sequence homology with other members of thetransferrin family (Metz-Boutigue et al. (1984) op.cit.; Pentecost etal. (1987) J. Biol. Chem. 262: 10134). A partial cDNA sequence forneutrophil hLF was published by Rado et al. (1987) Blood 70: 989), whichagrees by more than 98% sequence identity compared to the amino acidsequence determined by direct amino acid sequencing from hLF from humanmilk. The structures of the iron-saturated and iron-free forms of humanlactoferrin have been reported (Anderson et al. (1989) J. Mol. Biol.209: 711; Anderson et al. (1990) Nature 344: 784).

Protocols for purifying lactoferrin from milk have been reported. U.S.Pat. No. 4,436,658 describes the isolation of bovine lactoferrin fromdefatted and casein-free whey of bovine milk. Briefly, whey is contactedwith silica in a slightly basic medium at pH 7.7-8.8, the lactoferrin isadsorbed and thereafter eluted with 0.5M NaCl/0.1N acetic acid. U.S.Pat. No. 4,791,193 and European Patent Application No. EP 0 253 395 byOkonogi et al. similarly report a method wherein bovine milk iscontacted with carboxymethyl groups of a weakly acidic cation exchangeresin and the adsorbed lactoferrin is eluted with a 10 percent NaClgradient. In U.S. Pat. No. 4,668,771, bLF is isolated from bovine milkusing a monoclonal antibody fixed to an insoluble carrier. WO89/04608describes a process for obtaining fractions of bovine lactoperoxidaseand bLF from bovine milk serum; the milk serum is microfiltered andpassed through a strong cation exchanger, pH 6.5, at a high rate of flowfor selective adsorption of lactoperoxidase and bLF followed bysequential elution of lactoperoxidase with bLF with a 0.1-0.4M and a0.5-2.0M NaCl solution, respectively. U.S. Pat. No. 4,997,914 disclosesisolation of hLF from human colostrum or raw milk; thelactoferrin-containing sample is contacted with a sulfuric ester of acrosslinked polysaccharide to bind hLF, followed by elution with a0.4-1.5 NaCl aqueous solution.

The scientific literature also reports protocols for the isolation oflactoferrin from milk. A number of these involve isolation of LF from anatural source using ion-exchange chromatography followed by saltelution. Querinjean et al. (1971) Eur. J. Biochem. 20: 420, reportisolation of hLF from human milk on CM Sephadex C-50 followed by elutionwith 0.33M NaCl. Johannson (1969) Acta Chem. Scand. 23: 683 employed CMSephadex C-50 for purification of LF, and Johannson et al (1958) Nature181: 996 reports the use of calcium phosphate for LF purification.Torres et al. (1979) Biochem. Biophys. Acta 576: 385 report lactoferrinisolation from guinea pig milk. The milk was pre-treated bycentrifugation to remove fats and to sediment the casein. A WhatmanCM-52 column was used, and lactoferrin was eluted with 0.5M NaCl/5 mMsodium phosphate, pH 7.5. Roberts and Boursnell (1975) Jour. ofReproductive Fertility 42: 579, report lactoferrin isolated fromdefatted sow's milk. CM-Sephadex was added to an ammonium ferroussulfate precipitate of the milk, and the bound lactoferrin was elutedwith 0.5M NaCl/20 mM phosphate at pH 7 followed by a second CM-Sephadexfractionation from which the lactoferrin was eluted with 0.4M NaCl.Zagulski et al. (1979) Prace i Materialy Zootechniczne 20: 87, reportbovine lactoferrin isolated from bovine milk. Defatted bovine milk wasmixed with CM-Sephadex C-50, and lactoferrin was eluted from the columnwith 0.5M sodium chloride/0.02M sodium phosphate at pH 7. Moguilevsky etal. (1975) Biochem J. 229: 353, report lactoferrin isolated from humanmilk, using CM-Sephadex chromatography and elution with 1M sodiumchloride. Ekstrand and Bjorck (1986) Jour. of Chromatography 358: 429,report lactoferrin isolated from human colostrum and bovine milk.Defatted bovine or human milk was acidified, adjusted to pH 7.8 andapplied to a Mono S™ column. The bovine or human lactoferrin was elutedwith a continuous salt gradient of 0-1M NaCl. The purification of humanlactoferrin from bovine lactoferrin was not reported. Foley and Bates(1987) Anal. Biochem. 162: 296, report isolation of lactoferrin fromhuman colostrum whey. The whey was mixed with a weak ion-exchange resin(cellulose phosphate) and proteins were eluted with a stepped salt andpH gradient. Lactoferrin was eluted with 0.25M NaCl/0.2M sodiumphosphate at pH 7.5 . Further, Yoshida and Ye-Xiuyun (1991) J. DairySci. 74: 1439, disclosed the isolation of lactoferrin by ion exchange oncarboxymethyl cation resin using 0.05M phosphate buffer at pH 7.7 with alinear gradient of 0-0.55M NaCl. The carboxymethyl-Toyopearl columnadsorbed only lactoperoxidase and lactoferrin from the albumin fractionof bovine milk acid whey. Lactoferrin was eluted between 0.4-0.55M NaCland was separated into two components; lactoferrin A and lactoferrin B.

Other methods, including affinity chromatography, have also beenreported. For example, in Kawakami et al. (1987) J. Dairy Sci. 70: 752,affinity chromatography of LF with monoclonal antibodies to human orbovine lactoferrin was reported. Human lactoferrin was isolated fromhuman colostrum and bovine lactoferrin from bovine milk or cheese whey.(See also U.S. Pat. No. 4,668,771, cited supra) Hutchens et al. (1989)Clin. Chem. 35: 1928, lactoferrin was isolated from the urine of humanmilk fed preterm infants with single-stranded DNA on an affinity column.Additionally, Chen and Wang (1991) J. Food Sci. 56: 701 reported aprocess combining affinity chromatography with membrane filtration toisolate lactoferrin from bovine cheese whey using heparin-Sepharose tobind lactoferrin. Cheese whey was diluted with a binding buffer andadded to the heparin-Sepharose material. The slurry was microfiltered,and the lactoferrin was eluted with 5 mM veronal-hydrochloride/0.6M NaClat pH 7.4. Bezwoda et al. (1986) Clin. Chem. Acta 157: 89 report the useof Cibacron Blue F3GA resin for purification of LF. Ferritin (Pahud etal. (1976) Protides Biol Fluids. 23: 571) and heparin (Blackberg (1980)FEBS Lett. 109: 180) have also been reported for purification from milk.

Thus there exists a need in the art for methods for purification ofhuman lactoferrin from milk, particularly from milk of nonhumantransgenic animals, such as bovine species, that contain humanlactoferrin encoded by a transgene. It is one object of the invention toprovide methods and compositions for economical and efficientpurification of human lactoferrin from milk, such as bovine milk, foruse as a pharmaceutical or food additive. The present invention fulfillsthese and other needs. It is also an object of the present invention toprovide human lactoferrin compositions with a purity of about 98% orgreater.

The references discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

The present invention provides an efficient and effective procedure forpurification of human lactoferrin from milk, especially for purificationof human lactoferrin from bovine milk produced by transgenic bovinespecies containing a human lactoferrin transgene. The transgene-encodedhuman lactoferrin is substantially purified from other milk proteins inthe milk of transgenic cows, and is preferably substantially isolatedfrom endogenous bovine lactoferrin, if present in the milk.

The invention provides methods for isolating human lactoferrin,including human lactoferrin produced by expression of a transgeneencoding a recombinant human lactoferrin (rhLF), as well as otherrelated lactoferrin species from milk, typically from bovine milk. Suchother related lactoferrin species can include truncated hLF, amino acidsequence variants (muteins or polymorphic variants) of hLF, an hLFspecies which comprises additional residues. The invention also providesmethods that permit the purification of human lactoferrin (includingrhLF) from bovine lactoferrin, ovine lactoferrin, goat lactoferrin,mouse lactoferrin, and porcine lactoferrin. In general, milk or a milkfraction containing hLF is contacted with a strong cation exchange resin(e.g., S Sepharose™ sepharose) in the presence of relatively high ionicstrength (0.2M to 0.5M NaCl or KCl, preferably 0.4M NaCl or KCl) toprevent binding of non-lactoferrin proteins and other substances to thestrong cation exchange resin and to reduce electrostatic interactions oflactoferrin with other proteins (e.g., caseins) or substances (e.g.,lipopolysaccharide), and to liberate lactoferrin from complexes. Thestrong cation exchange resin containing the bound lactoferrin isseparated from the unbound compounds in the milk or milk fraction,typically by centrifugation or sedimentation followed by batchwisewashing and/or by pouring the resin into a column and washing the beadswith buffer having approximately equal or lower salt concentration. Thelactoferrin bound to the cation exchange resin is eluted with anaqueous, typically buffered, NaCl or KCl gradient (e.g., linear gradientof 0-1M NaCl in 20 mM sodium phosphate, pH 7.5 ) or by batch elution orstepwise elution with an aqueous, preferably buffered, NaCl or KClsolution of 0.4M or greater, preferably at least 0.5M NaCl or KCl. Byselecting appropriate elution conditions, human lactoferrin may besubstantially purified from bovine milk and substantially separated frombovine lactoferrin by an efficient method.

In one aspect of the invention, human lactoferrin (e.g., rhLF) isfurther purified from endogenous lactoferrin (e.g., bLF) by theadditional subsequent step of rechromatography on a strong cationexchanger, such as S Sepharose™ Fast Flow, with salt gradient orstepwise elution to separate human lactoferrin from remaining traces ofendogenous nonhuman lactoferrin species (e.g., bLF), and/or mayoptionally include affinity chromatography with a concanavalin A resinto further separate human lactoferrin from bLF, with bLF being morestrongly bound to the Con A resin than hLF.

In a method of the invention, a limiting quantity of a strong cationexchange resin (e.g., an amount less than that needed to saturably bindessentially all of the lactoferrin in the sample) is contacted to milkor a milk fraction (e.g.,. whey) under aqueous conditions (e.g., byadding resin directly to the milk or milk fraction) whereby thestrongest cationic proteins, such as lactoferrin, preferentially andcompetitively bind to the limiting amount of cation exchange resinpresent. With a limiting amount of cation exchange resin we thus mean aquantity that is just enough to at least bind essentially all (e.g., 99percent) molecules of an object lactoferrin species (e.g., human) at apredetermined salt strength of the milk or milk fraction. The amount ofstrong cation resin to be used is thus limited to optimize theselectivity of lactoferrin binding. The limiting amount of strong cationexchange resin with bound protein is separated from the remainder of themilk or milk fraction, typically by centrifugation or sedimentationfollowed by batchwise washing and/or pouring the resin into a columnfollowed by columnwise washing of the resin and bound proteins. Thelactoferrin bound to the resin is eluted by a high salt buffer (i.e.,NaCl or KCl concentration greater than 0.4M) or a salt gradient having amaximal salt concentration of at least about 0.5M NaCl or KCl).

In one variation, an ionic species, such as NaCl or KCl, is added to rawmilk, processed milk, or a milk fraction prior to contacting the milk ormilk fraction with a strong cation exchange resin. Typically, salt (or asalt solution) is added to the milk or milk fraction to bring the finalsalt concentration to approximately 0.2M to 0.5M, most preferably toapproximately 0.4M in NaCl or KCl, forming a high ionic strength milksolution. The high ionic strength milk solution is contacted with astrong cation exchange resin under binding conditions and the resincontaining bound milk protein(s) is separated from the unboundcomponents in the remainder of the high ionic strength milk solution,typically by centrifugation or sedimentation followed by batchwisewashing and/or by pouring the strong cation exchange resin mixture intoa column and removing the remainder of the high ionic strength milksolution by removing the liquid from the column and/or by washing theresin column with a wash buffer having an approximately equal or lowerionic strength than the high ionic strength milk solution. Thelactoferrin bound to the strong cation exchange resin is eluted by ahigh salt buffer (i.e., NaCl or KCl concentration greater than 0.4M) ora salt gradient having a maximal salt concentration of at least about0.5M NaCl or KCl). In an optional variation, a detergent, such as anonionic detergent (e.g., Tween-20) may be added to the milk or milkfraction to reduce undesirable hydrophobic interactions betweenmacromolecules that would reduce the efficiency of lactoferrinpurification. The milk or milk fraction also may be substantiallydiluted, typically to a final salt concentration of about 0.2M to 0.5MNaCl or KCl, preferably 0.4M NaCl or KCl, to further reduce undesiredintermolecular interactions that can reduce the yield and/or purity ofrecovered lactoferrin.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show the differential elution profiles of hLF (panel A) andbLF (panel B) from a strong cation exchange resin, Mono S™ by linearsalt gradient.

FIGS. 2A-2B show the elution profiles of hLF (panel A) and bLF (panel B)from a strong cation exchange resin, Mono S™, by stepwise elution.

FIGS. 3A-3B show resolution of hLF from bLF on a strong cation exchangeresin by linear salt gradient elution (panel A) and stepwise elution(panel B).

FIGS. 4A-4B show specific radioimmunoassays for hLF and bLF in theeluted fractions for linear salt gradient elution (panel A) and stepwiseelution (panel B).

FIGS. 5A-5B show the chromatographic resolution of hLF from bLF inhLF-spiked bovine milk (panel B) chromatographed on a Mono S™ columnwith linear salt gradient elution. Panel A shows (unspiked) controlbovine milk.

FIGS. 6A-6B show the chromatographic resolution of hLF from bLF inhLF-spiked bovine milk (panel B) chromatographed on a Mono S™ columnwith stepwise salt elution. Panel A shows (unspiked) control bovinemilk.

FIG. 7 shows the relationship between the amount of hLF bound to a MonoS™ column and the NaCl concentration at which elution of hLF wasobserved to begin when a linear salt gradient was applied.

FIGS. 8A-8B show profiles for the elution of hLF (A) and bLF (B) from aPhenyl Sepharose FF column.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

The term "naturally-occurring" as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

As used herein, "substantially pure" means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies.

As used herein, the term "enriched" refers to composition or fractionwherein an object species has been partially purified such that, on amolar ratio basis, at least about 10 percent of one or more naturallyoccurring contaminant species have been removed. For example, a samplefrom milk of a transgenic bovine expressing human lactoferrin may beenriched for human lactoferrin by selectively removing caseins by acidprecipitation (e.g., the whey fraction is thereby enriched for humanlactoferrin).

As used herein, "human lactoferrin" comprises a polypeptide having anamino acid sequence substantially as described by Metz-Boutigue et al.(1984) Eur. J. Biochem. 1451: 659, noting the sequence inconsistenciesidentified in PCT publication WO91/08216 and other published protein andDNA sequences. The term human lactoferrin also includes naturallyoccurring human allelic variants either (partially) proteolyzed or not("naturally occuring human lactoferrin") and amino acid sequencevariants that have been modified by the insertion, substitution, ordeletion of one or more amino acids as compared to a naturally occurringhuman lactoferrin species and which have a greater degree of sequenceidentity when optimally aligned (and gapped, if necessary) to anaturally occurring human lactoferrin amino acid sequence of at least 50contiguous amino acids than to other naturally occurring polypeptidespecies of at least 50 contiguous amino acids. Human lactoferrin alsoincludes recombinantly encoded human lactoferrin ("rhLF") expressed in atransgenic nonhuman animal, such as a bovine, where the glycosylationpattern may be distinct from glycosylation patterns of naturallyoccuring human lactoferrin obtained from human milk.

DETAILED DESCRIPTION

Human lactoferrin may be used for pharmaceutical uses (WO91/13629,incorporated herein by reference), as a nutrient supplement, and forother uses. For such uses it is frequently necessary or preferable toemploy human lactoferrin which has been purified, either partially oressentially to homogeneity, from undesired contaminants in milk,especially from other milk proteins (e.g., whey proteins, caseins), milkfat, and other contaminants (e.g., lipopolysaccharide of Gram-negativebacteria) present in milk samples. Lactoferrins have been reported tointeract with a wide variety of milk proteins including IgA, caseins,SC, albumin, lysozyme, β-lactoglobulin, and others. The presentinvention provides purification methods which advantageously afford theefficient and rapid purification of lactoferrin, especially humanlactoferrin, from milk, such as milk produced by transgenic bovinespecies harboring a human lactoferrin transgene which is expressed inthe mammary gland.

A basis of the invention is the finding that lactoferrin, especiallyhuman lactoferrin, has a surprisingly strong affinity for strong cationexchange resins which can be exploited to purify lactoferrin from milk,especially to purify human lactoferrin from milk of transgenic bovinespecies expressing a human lactoferrin transgene. It has also beendiscovered that elevating the ionic strength of milk or a milk fractionto approximately 0.2-0.5M NaCl or KCl or equivalent salt, preferablyapproximately 0.35-0.4M NaCl or KCl, more preferably approximately 0.4MNaCl or KCl, and typically 0.4M NaCl concomitant with contacting themilk or milk fraction with the strong cation exchange resin(s) enhancesrecovery and resolution of lactoferrin from undesired contaminants inthe milk or milk fraction. The addition of relatively high saltconditions (e.g., to a final concentration of about 0.4M NaCl) to milkor milk fractions strongly reduces binding of most contaminant proteins(e.g., whey proteins and caseins) and lipopolysaccharides ("LPS") to astrong cation exchange resin (e.g., Mono S™ or S Sepharose™ Fast Flow)while permitting efficient binding of lactoferrin (e.g., rhLF) to thestrong cation exchange resin, thus providing a convenient basis forseparation of lactoferrin from contaminant molecular species. It isnoted that elevating the ionic strength of milk or milk fractions toapproximately 0.35-0.4M NaCl virtually excludes the binding of any otherbovine milk protein other than lactoferrin to a strong cation exchangeresin. The elution profile of bovine whey that has been applied to MonoS™ under conditions of low ionic strength shows that all bound bovinewhey proteins (except lactoferrin) elute at NaCl concentrations of 0.3MNaCl (e.g., bovine lactoperoxidase) or lower (other proteins). Moreover,it has also been found that strong cation exchange resins and relativelyhigh salt conditions may be used to separate human lactoferrin frombovine lactoferrin in milk or milk fractions. Elevated salt conditions(NaCl concentration at least 10 mM greater than physiological milk,generally 0.2M NaCl or greater) are used to enhance specific loading ofhuman lactoferrin onto strong cation exchange resins. Elevated ionicstrength milk or milk fractions exhibit more selective binding of hLF tothe strong cation exchange resin selected.

In a preferred embodiment, human lactoferrin is expressed and secretedinto the milk of a transgenic animal, preferably a bovine species. Inthose embodiments where rhLF is expressed and secreted into the milk oftransgenic bovine species, the transgenic milk may be either used asobtained or further treated to purify the rhLF. The human lactoferrinobtained by the methods of the invention preferably is obtained byprocessing a milk fraction, although raw milk (whole milk) may also beused. Preferable milk fractions include defatted milk, defatted milkfrom which particulate matter and/or caseins have been removed (e.g.,milk whey), and other milk fractions which contain lactoferrin.

Preparation of Milk and Milk Fractions

Raw milk is harvested from the transgenic nonhuman animal expressinghuman lactoferrin. The raw milk (whole milk) may optionally be adjustedto a relatively high ionic strength (e.g., 0.3-0.4M NaCl or KCl) byaddition of solid NaCl, KCl, or other suitable salt, or an aqueoussolution thereof. Combinations of monovalent salts may be used (e.g.,NaCl and KCl together), if desired, so that the final concentration ofmonovalent cation in the milk will be approximately 0.3-0.4M. The wholemilk may be contacted with a strong cation exchange resin directly, ormay be processed into a milk fraction which is subsequently contactedwith a strong cation exchange resin under conditions of relatively highionic strength (e.g., 0.3-0.4M NaCl or KCl). If the whole milk is to becontacted with a strong cation exchange resin directly, the ionicstrength is adjusted by increasing the salt concentration in the wholemilk, typically to approximately 0.35-0.4M NaCl or KCl, prior to orconcomitant with contacting the whole milk with the strong cationexchange resin. Optionally, the whole milk may be diluted with anaqueous solution, typically a buffered salt solution, to produce dilutedwhole milk having a monovalent cation (e.g., Na⁺, K⁺, or combinationthereof) concentration of approximately 0.3-0.4M. Usually a bufferedsalt solution used as a whole milk diluent will have a NaCl (or KCl)concentration of at least about 0.3-0.4M and will be buffered to pH 6-8with a suitable buffer; the final concentration of monovalent cation(s)in the diluted milk is preferably 0.3-0.4M, more preferably 0.4M, andthus cationic species contributed by a buffer, such as a sodiumphosphate buffer, should be taken into account along with the dissolvedsalt species (e.g., NaCl and KCl). Thus, high ionic strength milk (i.e.,milk having at least approximately 0.3-0.4M monovalent cation) may begenerated by either adding and dissolving one or more solid salts (e.g.NaCl or KCl) in whole milk or by diluting whole milk in a diluent saltsolution whereby the final concentration of monovalent cation in thediluted milk is approximately 0.3-0.4M. High ionic strength whole milk(diluted or undiluted) may be used directly for contacting with a strongcation exchange resin, or may be processed further into a milk fractionprior to contacting with a strong cation exchange resin. Whole milkwhich has not had the ionic strength increased by the addition of salttypically is processed into a milk fraction, the ionic strength of themilk fraction is then increased by addition of one or more salts toincrease the concentration of monovalent cation to approximately0.3-0.4M, and the high ionic strength milk fraction is contacted with astrong cation exchange resin.

Without wishing to be bound by any particular theory, dilution of wholemilk may also decrease undesired intermolecular interactions betweenlactoferrin (e.g., rhLF) and contaminant macromolecular species (e.g.,caseins, LPS of Gram-negative bacteria) present in milk. However,significant dilution of whole milk or milk fractions may result in largevolumes which may be less efficiently processed. It has been found thatincreasing the ionic strength of the milk also substantially decreasesundesired intermolecular interactions between lactoferrin (e.g., rhLF)and contaminant macromolecular species, permitting a more facilepurification without necessitating dilution of milk to large volumes,unless desired. Optionally, addition of a non-interfering detergent(i.e., does not substantially reduce binding of hLF to resin), such as anonionic surfactant (e.g., Tween-20), to a concentration ofapproximately 0.001-0.2 percent, preferably about 0.01-0.03 percent, mayalso contribute to reducing undesired intermolecular interactionsinvolving lactoferrin.

Processing of whole milk, either directly or after increasing the ionicstrength by addition of salt (e.g., NaCl or KCl), into a milk fractioncontaining lactoferrin prior to contacting with a strong cation exchangeresin may further reduce the amount of one or more contaminant species.For example, defatting of whole milk can remove a significant proportionof lipid species which may be undesirable and which may interfere withefficient purification of lactoferrin by strong cation exchangechromatography.

The human lactoferrin obtained by the process of the invention ispreferably obtained by processing transgenic milk whey. Milk whey is amilk fraction produced by removing substantially all fats and caseinsfrom the transgenic milk. A variety of methods (e.g., centrifugation)are known to those skilled in the art to remove fats from milk.Similarly, a number of procedures are known to those skilled in the artfor removal of caseins from milk. For example, acid precipitation orproteolysis of kappa-casein by chymosin may be used to remove caseinfrom milk. Other compatible methods for generating milk fractionscontaining lactoferrin (e.g., whey) known to those skilled in the artmay be used. Although salt may be added subsequent to the removal ofcaseins from the milk, that order of addition may result in the loss ofsignificant amounts of lactoferrin, as caseins are generally highlyphosphorylated (i.e., negatively charged) and may bind substantialamounts of lactoferrin, presumably by electrostatic interactions; thus,removal of caseins from milk under low salt conditions may result in theundesirable removal of substantial amounts of lactoferrin as well.

In a preferred embodiment, salt (e.g., NaCl or KCl) is added to wholemilk of a transgenic nonhuman animal expressing human lactoferrin beforedefatting and removal of caseins; alternatively, salt may be added afterdefatting but prior to removal of caseins. Typically, an aqueoussolution containing NaCl and a sodium phosphate buffer is used to dilutewhole milk to form high ionic strength milk having a final concentrationof 10-50 mM sodium phosphate, 0.3-0.4M NaCl at about pH 6.5-7.5. Thehigh ionic strength milk typically has a final concentration of 20 mMsodium phosphate, 0.4M NaCl at about pH 7.0. Optionally, a surfactantmay be included to a final concentration of about 0.001-0.2 percent byvolume, with a typical concentration being approximately 0.02 percentv/v. Usually, a nonionic surfactant such as Tween-20 is used, othernonionic surfactants may also be suitable. Thus, in this embodiment,defatted milk containing approximately 0.4M NaCl, and optionallycontaining about 0.02 percent Tween 20, is produced and subsequentlyused for removal of caseins by conventional methods to produce a highionic strength whey containing lactoferrin. After the foregoing step,the high ionic strength whey is contacted with a strong cation exchangeresin (e.g., S Sepharose™ Fast Flow, Pharmacia Biotechnology,Piscataway, N.J.) under suitable binding conditions whereby the strongcation exchange resin preferentially binds lactoferrin from the highionic strength whey to produce a lactoferrin-resin complex.

Binding of Lactoferrin to Strong Cation Exchange Resins

Human lactoferrin (calculated pI approximately 9.7) essentially does notbind to the strong anion exchange resin Mono Q™ at pH 4.5 and weaklybinds at pH 7.5 (i.e., even at pH values below its pI), which isconsistent with the idea that charge is unevenly distributed over thesurface of the human lactoferrin molecule.

However, human lactoferrin binds almost completely to strong cationexchange resins, such as Mono S™ or S Sepharose™, between approximatelypH 4.5 and 9.5 and at monovalent cation concentrations equivalent toapproximately 0.5M NaCl or less, preferably 0.45M NaCl or less. Thisfinding is consistent with a hypothesis that human lactoferrin behavesas a molecular dipole, possibly because of the many basic amino acids(Arg and Lys) clustered together in the amino-terminal portion. Thisfinding provides a basis for purification of human lactoferrin from milkby reversible adsorption to a strong cation exchange resin underconditions of relatively high salt.

A multitude of strong cation exchange resins are known to those of skillin the art of protein purification. "Strong cation exchange resins" aredefined as those which exchange their diffusible cation over a wide pHrange, such as those containing sulphite or sulphate groups. Presentlypreferred strong cation exchange resins are, for example, Mono S™ cationexchange resin and S Sepharose™ Fast Flow cation exchange resin(available from Pharmacia/Pharmacia LKB Biotechnology, Piscataway,N.J.). The Mono S™ and S Sepharose™ Fast Flow cation exchange resinscurrently use ligand S, although ligand SP is suitable as well and maybe substituted. Resins with similarly charged groups providing strongcation exchange will also be useful for purposes of the invention.

A variety of methods may be used to contact the milk or milk fraction,including transgenic bovine whey containing human lactoferrin with astrong cation exchange resin. For example, the strong cation exchangeresin may be formed into a column and the lactoferrin-containing wheymay be passed through the column. After adsorption of the lactoferrin tothe column resin, the column is washed and the human lactoferrin issubsequently desorbed by elution at an ionic strength sufficient toelute the human lactoferrin, preferably at an ionic strength sufficientto elute human lactoferrin efficiently but not substantially elutebovine lactoferrin, if present. Alternatively, thelactoferrin-containing whey may be contacted with a strong cationexchange resin in the form of a bed, generally under conditions ofrelatively high ionic strength (e.g., 0.35-0.4M NaCl, pH 7.0). In thiscase, the lactoferrin-containing whey is contacted with the strongcation exchange resin in a bed and agitated for a suitable period oftime at a suitable temperature (e.g., 4-30 degrees Celsius); suchcontacting may be accomplished by mixing resin beads directly into thelactoferrin-containing whey. Subsequently, the strong cation exchangeresin with the adsorbed lactoferrin is separated by, for example,centrifugation, spontaneous sedimentation, or by being formed into acolumn, and the mobile phase (i.e., lactoferrin-depleted whey)substantially removed.

The isolated lactoferrin-resin complexes can be washed to removeremaining unbound or resin-bound non-lactoferrin species, if desired,with a wash buffer having an ionic strength sufficiently low (e.g.,monovalent cation concentration less than about 0.45M) to preventsubstantial elution of lactoferrin from the resin, but preferablysufficiently high to elute non-lactoferrin macromolecules which may ormay not be bound to the resin. Typically, a wash buffer can comprisebetween about 0.01-0.45M NaCl, 5-50 mM sodium phosphate buffer atapproximately pH 7.5, although other wash solutions may be used. Forexample, a wash buffer containing 0.3M NaCl, 10 mM sodium phosphate pH7.5 may be used. Optionally, a detergent such as a nonionic surfactant(e.g., Tween-20) may be included in a wash buffer, typically at 0.01-0.1percent by volume. The isolated lactoferrin-resin complexes may bewashed in a single washing step, in multiple washing steps, or mayremain unwashed. Generally, at least one volume of wash buffer per unitvolume of resin is used to wash the isolated lactoferrin-resincomplexes, frequently larger wash volumes are used, especially if thewash buffer has low ionic strength (e.g., less than 0.2M NaCl), ifwashing is performed. Washing serves to remove non-lactoferrinmacromolecules from the resin whether in column or bed form.

Lactoferrin is selectively desorbed from the isolated lactoferrin-resincomplexes by elution with a solution of appropriate ionic strength.Generally, an elution buffer will comprise a monovalent salt (e.g., NaClor KCl) at a concentration of at least approximately 0.3-0.4M, typically0.45-0.5M or greater, and preferably also contains a suitable buffer,such as sodium phosphate (e.g., 5-50 mM) at a pH of approximately 7.5. Avariety of salt concentrations and compositions of the elution solutionare possible, and may vary with the scale of purification, so thatcalibration or standardization of column or resin bed performance may beperformed by those skilled in the art using standard calibrationmethods. Alternatively, human lactoferrin can be selectively desorbedfrom the lactoferrin-resin complexes packed in a column by elution withan NaCl or KCl gradient or step gradient as the mobile phase, withdesorption of human lactoferrin typically occurring at approximately0.30-0.75M NaCl, depending upon the scale of purification and othercolumn performance factors. Calibration of column performance anddetermination of elution profile can be readily determined by those ofskill in the art for each embodiment. The fraction(s) containing humanlactoferrin may be identified by any convenient assay for humanlactoferrin, including but not limited to: immunoassay using antibodywhich specifically binds human lactoferrin (Nuijens et al. (1992) J.Lab. Clin. Med. 119: 159) or other assays for detecting and quantitatinglactoferrin.

In a further embodiment, lactoferrin is removed from lactoferrin-resincomplexes by dialysis. Dialysis is a widely practiced technique known tothose of skill in the art. The dialysis membrane is selected such thatthe protein, for example human lactoferrin, passes unimpeded from oneside of the membrane to the other. In the present case, a strong cationexchange resin is present on the side of the membrane opposite the sidecontacting the transgenic milk or milk fraction containing lactoferrin.Lactoferrins, including human and bovine lactoferrin, partition from themilk or milk fraction across the dialysis membrane to the sidecontaining the strong cation exchange resin, where they bind the strongcation exchange resin. The pore size (molecular weight cutoff) of thedialysis membrane selected prevents the lactoferrin-resin complexes frompassing across the membrane into the milk or milk fraction. In thisembodiment, the dialysis membrane is selected so that only the unboundlactoferrin, and not the lactoferrin bound to the strong cation exchangeresin, passes across the membrane. Typically, the milk or milk fractionis adjusted to an ionic strength of about 0.35-0.4M monovalent salt(NaCl or KCl) and is generally buffered to a pH of about 7.5, usuallywith a suitable buffer (e.g., sodium phosphate, potassium phosphate)prior to or concomitant to contacting the milk or milk fraction with thedialysis membrane separating the strong cation exchange resin from themilk or milk fraction. Optionally, a detergent, such as a nonionicsurfactant (e.g., Tween-20), may be included at a concentration ofapproximately 0.001 to 0.2 percent v/v, preferably about 0.02 percentv/v, if present. After a suitable dialysis period for allowing partitionof lactoferrin across the membrane to bind to the strong cation exchangeresin, the milk or milk fraction thus depleted of lactoferrin is removedand the lactoferrin bound to the strong cation exchange resin is eluted,either directly or across a dialysis membrane, by contacting thelactoferrin-resin complexes with a high ionic strength buffer (e.g.,0.45-0.7M NaCl or KCl, 5-50 mM sodium phosphate or potassium phosphatepH 7.5). A variety of alternate aqueous solutions may be used to elutethe lactoferrin from the strong cation exchange resin and will beapparent to those of skill in the art. Substitution of other salts(e.g., LiCl), including salts of divalent cations may also be employedby those of skill in the art to practice the invention.

Lactoferrin eluted from the lactoferrin-resin complexes may be subjectedto an additional round of cation exchange chromatography and/or lectin(e.g., Con A) affinity chromatography to resolve further the humanlactoferrin from bovine lactoferrin, if present. Moreover,rechromatography may be used to purify further intact human lactoferrinfrom degradation (proteolyzed) products. Preferably, intact humanlactoferrin is recovered in substantially pure form.

After eluting the lactoferrin from the bound lactoferrin-resin complexeswith a high ionic strength salt solution, the eluted lactoferrin may betreated to clarify and concentrate it. Preferably the recoveredlactoferrin is dialyzed to remove salts, ultrafiltered, and optionallylyophilized to concentrate the lactoferrin. Suitable storage and/orreconstitution components (e.g., buffers, salts, preservatives,antibiologicals, ferric ions) may be added to the purified lactoferrin.

Purification of Lactoferrin from Milk by Batch Extraction

Batch extraction of lactoferrin from milk using a strong cation exchangeresin is believed to be a preferred purification strategy for largescale industrial applications.

In batch extraction, it is generally preferable that salt is added tothe milk or milk fraction to produce a high ionic strength milk solution(e.g., 0.4M NaCl) prior to, or concomitant with, contacting the strongcation exchange resin with the milk solution. It is believed that saltis added to increase the ionic strength sufficiently to (1) reducepotential intermolecular electrostatic interactions between contaminants(e.g., caseins and LPS) and lactoferrin, (2) reduce binding ofnon-lactoferrin macromolecules to the strong cation exchange resin,i.e., to secure selectivity of lactoferrin binding in case anon-limiting amount of a strong cation exchange resin (protein-bindingcapacity in excess of the amount of object lactoferrin species) is addedto milk or milk fraction, (3) induce aggregation of certainnon-lactoferrin species which may then be removed before a column isprepared, and (4) prevent aggregation of materials (possibly includinglactoferrin) that may be retained on the resin in low salt conditionsand become captured in the chromatography column when a high ionicstrength wash or salt gradient is applied, thus impeding columnperformance and allowing partial disaggregation and elution of theaggregate to contaminate eluted lactoferrin. However, the ionic strengthshould not be excessively high so that lactoferrin is not efficientlybound by the strong cation exchange resin. For example, an ionicstrength of approximately 0.35-0.45M NaCl is generally preferred, with0.4M NaCl typically used.

Further, when lactoferrin is extracted from milk fractions such as whey(e.g., prepared by acid precipitation, chymosin treatment, orultracentrifugation), the recovery of lactoferrin is increased if salthas been added to increase the ionic strength prior to casein removal.This effect is presumably due to the trapping of lactoferrin byelectrostatic interaction with caseins and may be overcome by increasingthe ionic strength prior to casein removal.

For example and not limitation, milk from a transgenic nonhuman animalexpressing rhLF may be processed according to the following protocol forsubstantially purifying the human lactoferrin from the milk.

First, solid NaCl or a 5M NaCl stock solution is added to the milk toproduce a final concentration of 0.35-0.45M NaCl, usually about 0.4MNaCl. Optionally, a nonionic surfactant (Tween-20) is added up to a 0.02percent (v/v) final concentration. Sodium phosphate is optionally addedto a final concentration of about 20 mM with an expected pH of about7.5, for example by adding NaH₂ PO₄ H₂ O and Na₂ HPO₄ 2H₂ O up to afinal concentration of 3.2 mM and 16.8 mM, respectively; the final pH ofthe milk solution need not be exactly 7.5, and frequently will beapproximately 6.5-7.0. It is believed that the addition of sodiumphosphate (or other buffering agent) may be omitted, as hLF bindsefficiently to strong cation exchange resins (e.g., Mono-S™ or SSepharose™) at pH values between 4.5 and 9.5. Milk fat is removed bycentrifugation at 1600×g for 10 minutes in 500 ml polyallomer tubes in aBeckman JA-10 rotor to produce high ionic strength skim milk.Alternatively, spontaneous sedimentation followed by physical removal ofthe fat layer may be used. Alternatively, milk fat may be removed bycentrifugation after batchwise incubation of processed milk with astrong cation exchange resin. A strong cation exchange resin (e.g., SSepharose™, Pharmacia LKB, Piscataway, N.J.) ) typically is equilibratedwith high ionic strength buffer (0.4M NaCl, 20 mM sodium phosphate, pH7.5, optionally including 0.02 percent Tween-20) and dissolved in theprocessed milk. Approximately 1 ml of packed, equilibrated strong cationexchange resin beads equilibrated with high ionic strength buffer areadded per 5-20 mg of lactoferrin in the processed milk sample (which maybe determined by lactoferrin assay) and stirred for a suitable mixingperiod (e.g., about 1 hour or more, preferably overnight) to bind thelactoferrin to the resin beads. The resin beads (e.g., Fast-S™) are thenpelleted by centrifugation at approximately 1600×g for 5 minutes and thesupernatant is removed. Alternatively, beads may be pelleted byspontaneous sedimentation after which either high ionic strength skimmedor whole milk depleted of lactoferrin is removed. The pelleted resinbeads are washed three times with approximately one volume of high ionicstrength buffer, and the resin beads with one volume of washing bufferare then poured into a column. A substantially purified preparation oflactoferrin is eluted from the column with a gradient (i.e., 1.25 timesthe column volume) of 0.4-1.0M NaCl in 20 mM sodium phosphate pH 7.5.Excess salt present in the recovered lactoferrin can be removed bydialysis against saline or phosphate buffered saline (dialysis againstdistilled water tends to cause precipitation of lactoferrin). Humanlactoferrin can be separated from endogenous (nonhuman; e.g., bovine)lactoferrin by eluting the column with a salt gradient or stepwiseelution, with native human lactoferrin (e.g., rhLF) eluting from thecolumn at a lower or higher ionic strength than nonhuman (e.g., bovineor porcine, respectively) lactoferrin at pH 9.5 or lower. For example,when using a buffer of 20 mM sodium phosphate pH 7.5, rhLF typicallyelutes at a NaCl concentration that is approximately 50-100 mM(typically 70 mM) lower than the NaCl concentration at which efficientelution of bLF occurs. If additional purification of rhLF from bLF isdesired, the recovered fractions containing substantial amounts of rhLFmay be rechromatographed on a strong cation exchange resin and/or ConAcolumn and eluted with a salt gradient or stepwise elution to resolvefurther the hLF from bLF in the eluted fractions.

Lactoferrin Formulations

Lactoferrin produced by the methods of the invention is substantiallypurified. That is, the lactoferrin is substantially free fromcontamination with other milk proteins and molecular contaminantspresent in milk, including bacterial lipopolysaccharides. Humanlactoferrin produced by the methods of the invention is suitable forformulation in pharmaceutical or nutrient supplements comprisinglactoferrin, typically comprising from at least about 1 milligram toseveral grams or more of lactoferrin per dose. In view of theantibacterial and anti-inflammatory properties of human lactoferrin, avariety of uses for the purified human lactoferrin are possible. Forexample, antibacterial pharmaceuticals and/or nutritional supplementscomprising lactoferrin, especially human lactoferrin, may be producedand administered, typically in conjunction with other agents, to apatient for therapy or prophylaxis of local infection, large scale(bacterial) infection, blood-borne infection (sepsis) as well asinflammation resulting from an infection or non-infectious inflammatorydiseases (e.g., chronic inflammatory disease of ileum or colon). Suchpharmaceutical compositions are prepared containing lactoferrin,preferably human lactoferrin, in combination with a physiologicallycompatible carrier, typically for administration as an aqueous solution(e.g., in physiological saline) by intravenous, intramuscular,subcutaneous, topical, gavage, lavage, or other suitable routes ofadministration.

Similarly, pharmaceutical preparations and nutritionals containingsubstantially purified lactoferrin can be used, typically in conjunctionwith other pharmaceuticals or nutritionals, to treat large scalebacterial infections. For example, pharmaceutical preparationscontaining human lactoferrin can be used to treat blood-borne bacterialinfections, either alone or in conjunction with another pharmaceuticalagent, or are used to prepare or treat organ transplant recipients orother immunosuppressed individuals (e.g., AIDS patients) against theeffects of infections.

Additionally, the substantially purified human lactoferrin can be usedin the formulation of nutritional supplements. For example, for humanuse, the purified human lactoferrin can be included in infant formulasor be used as an adult dietary supplement. As a result of lactoferrin'siron-binding properties, nutritional preparations useful for treatingiron deficiencies (e.g., iron deficiency anemia of premature infants)may be formulated with substantially purified lactoferrin.

Quality Control Assays for Lactoferrin

Quality control of purified hLF should include at least one, preferablymore, optionally all of the following procedures: (1) non-reduced andreduced SDS-PAGE, with samples loaded without prior boiling and afterboiling, (2) spectroscopic analysis such as absorption measurement at280 and 465 nm, (3) radioimmunoassay analysis of hLF and bLF, (4) strongcation exchange chromatography (e.g., Mono S™), and (5) N-terminalprotein sequencing.

The following examples are offered for illustration and not to limit theinvention.

EXPERIMENTAL EXAMPLES Effect of Ionic Strength on Lactoferrin Recovery

We have found that the addition of salt to transgenic mouse and bovinemilk before caseins are removed very significantly increases the yieldof transgenic hLF and bLF, respectively, upon purification from wheyfraction. The data presented in Table 1 provide the background for thesedifferential recoveries.

                  TABLE 1    ______________________________________    % of total bLF                                     acid precipitation/               only centrifugation   centrifugation    Samples    no addition                         +0.4M NaCL  no addition    ______________________________________    Whole cow milk               100 + 13  100 + 13    100 + 11    Casein fraction                21 + 6.sup.a                         4 + 3       .sup. 32 + 10.sup.b    Whey fraction               83 + 6    99 + 11     72 + 10    ______________________________________     bLF was determined by competitive inhibition RIA in samples of whole     bovine milk (n = 10) as well as in whey and casein fractions prepared     after centrifugation for 30 min at 10,000 g. Acid precipitation was     performed by adjusting whole milk to pH 4.6, followed by incubating the     milk for 30 min at 40° C. Results (mean + SD) are expressed as     percentage of total bLF found in respective fractions.     .sup.a -bLF concentration in the casein pellets was on the average 4.0     times higher than in the whey fraction.     .sup.b -bLF concentration in the casein pellets was on the average 8.0     times higher than in the whey fraction.

bLF was determined by competitive inhibition RIA in samples of wholebovine milk (n=10) as well as in whey and casein fractions preparedafter centrifugation for 30 min at 10,000 g. Acid precipitation wasperformed by adjusting whole milk to pH 4.6, followed by incubating themilk for 30 min at 40° C. Results (mean+SD) are expressed as percentageof total bLF found in respective fractions. ^(a) -bLF concentration inthe casein pellets was on the average 4.0 times higher than in the wheyfraction. ^(b) -bLF concentration in the casein pellets was on theaverage 8.0 times higher than in the whey fraction.

Effect of Ionic Strength and Deterrent on Human Lactoferrin Binding

The effects of salt concentration and the presence of nonionicsurfactant (Tween-20) or cationic surfactant (Polybrene) on binding ofhuman lactoferrin (hLF) labeled with ¹²⁵ I to various ligandsimmobilized on Sepharose were determined. Sepharoses were suspended in10 mM sodium phosphate, 0.15M NaCl, pH 7.4. The following ligands wereimmobilized on Sepharose and used for determination of binding to hLF:R595, an LPS from S. minnesota (KDO⁺, no O antigen, rough); J5, an LPSfrom E. coli (KDO⁺, no O antigen, rough); heparin, a polyanion known tobind lactoferrin; HSA, human serum albumin; and glycine. ¹²⁵ I-labelledhLF was contacted with the Sepharose-ligand resins and exposed tovarious combinations of NaCl concentration and detergent. Table 2 showsthe percentage of ¹²⁵ I-hLF (i.e., radioactivity) retained on thevarious Sepharose-ligand resins.

                  TABLE 2    ______________________________________    Percentage Binding of .sup.125 -I-HLF to Various Sepharoses               Ligand Immobilized on Sepharose    Addition     R595   J5      Glycine                                      Heparin HSA    ______________________________________    no           68     57      5     68      8    +0.2% Tw     70     57      5     65      9    +0.2% Tw     10      7      3     35      4    +0.25 M NaCL    +0.2% Tw      5      5      2      5      3    +0.5 M NaCL    +0.2% Tw      4      4      1      2      2    +1 M NaCL    +0.2% Tw      3      4      1      3      4    +2 M NaCL    +0.2% Tw     18     13      3      6      4    +0.1% Polybrene    ______________________________________

Table 2 indicates that ionic strength of greater than about 0.25M NaCland detergent concentration of 0.2% Tween-20 substantially reducesbinding of hLF to various ligand species, including bacterial LPS. Thisexperiment indicates that the interaction between LPS and hLF is of anelectrostatic nature.

Studies were performed to determine the binding of hLF to LPS types froma wide variety (i.e., more than 50 types) of clinically relevantGram-negative bacteria. Lactoferrin reacted with varying affinities toeach type of LPS evaluated. Lactoferrin appeared to electrostaticallyinteract with the lipid A moiety of LPS, to a site identical or in closeproximity to the site of interaction of LPS with Polymyxin B, anantibiotic known to neutralize the toxic effects of LPS.

Elution Pattern of Lactoferrin from Ion Exchange Resins

Fifty micrograms of protein (hLF Sigma 1--see legend of Table 5--andbLF, Sigma, St. Louis, Mo.) in various buffered solutions of differentpH was applied to Pharmacia HR5/5 columns containing 1 ml of Mono S™(strong cation exchanger) or Mono Q™ (strong anion exchanger) resinequilibrated in appropriate buffers using the FPLC™ system (Pharmacia,Piscataway, N.J.) for determination of elution profiles with NaCl invarious buffers of various pH (sodium succinate, sodium acetate, sodiumphosphate, Hepes, Tris, ethanolamine, N-methylpiperidine). After washingeach column with 2 ml of the buffer, a linear salt gradient from 0-1.0MNaCl in 30 ml of buffer was applied at a flow rate of 1 ml/min. Peakswere monitored by absorption measurement at 280 nm. The elution of hLFand bLF in various buffer systems are shown in Table 3.

                                      TABLE 3    __________________________________________________________________________    Elution pattern of LF preparations on Mono S ™ and MonoQ ™ columns                        NaCl concentration (M)    Sample              at which a peak is eluted.    (FPLC run no.)            Buffer      (% of total hLF eluting at this position)    __________________________________________________________________________    Mono S ™    hLF(330)            50 mM Na succ pH 4.5                        0.56 (18.2)                              0.63 (44.3)                                     0.69 (37.1)    hLF(311)            50 mM Na acet pH 5.0                        0.57 (18)                              0.65 (42.4)                                     0.72 (38.4)    hLF(309)            20 mM Na phos pH 7.5                        0.51 (20.1)                              0.59 (46.3)                                     0.68 (33.8)    hLF(306)            50 mM Hepes pH 7.5                        0.51 (21.5)                              0.60 (43)                                     0.69 (34.7)    hLF(352)            20 mM Tris pH 7.5                        0.53 (29.4)                              0.63 (48.2)                                     0.71 (16.5)    hLF(278)            20 mM Tris pH 7.5                        0.52 (23.2)                              0.61 (50.8)                                     0.71 (25.5)    bLF(280)            20 mM Tris pH 7.5        0.78 (100)    hLF(355).sup.b            50 mM Hepes pH 8.0                        0.45 (23.1)                              0.53 (42.3)                                     0.64 (20.1)    hLF(350).sup.a            20 mM eth am pH 9.5                        0.43 (23.5)                              0.51 (39.5)                                     0.60 (15)    Mono Q    hLF(368)            20 mM N-meth pip pH 4.5                              no binding    hLF(360)            20 mM Tris pH 7.5  0.11    bLF(363)            20 mM Tris pH 7.5 0-0.23    hLF(365)            20 mM eth am pH 9.5                               0.16    __________________________________________________________________________     .sup.a about 21% of the hLF applied did not bind to the column.     .sup.b about 14% of the hLF applied did not bind to the column.

Table 3 shows that hLF binds nearly completely to Mono S™ between pH 4.5and 9.5, indicating that basic amino acid residues are clustered in hLF.Indeed, the N-terminus of hLF contains clustered basic amino acidresidues and thus hLF appears as a macromolecular dipole. hLF elutes inthree peaks, in contrast to the literature data. Reduced and nonreducedSDS-PAGE analysis of hLF peaks I, II, and III (see Tables 2 and 4)revealed no cleavage of hLF. All peaks contained the same proportion ofFe-hLF (non-reduced, nonboiled SDS-PAGE). The amount of hLF detected byRIA corresponds to the chromatogram.

hLF does not bind to Mono Q™ at pH 4.5 and binds weakly at pH 7.5 (pHvalue below its pI). This correlates with the idea that charge isunevenly distributed over the hLF molecule. On the basis of thesefindings, we selected a strong cation exchange resin at approximatelyneutral pH for purification of hLF.

Batch Purification of Lactoferrin from Milk

Lactoferrins were purified from human milk, bovine milk, and milk fromtransgenic mice expressing rhLF in their milk by the batch extractionmethod. Solid NaCl was added to a final concentration of 0.4M andTween-20 was added to a final concentration of 0.02% (v/v). Sodiumphosphate buffer (pH 7.5) was added to 20 mM final concentration butfinal pH was not adjusted to 7.5. Milk fat was removed by centrifugationfor 10 minutes at 1600×g in 500 ml polyallomer tubes in a Beckman JA-10rotor. Packed S Sepharose™ Fast Flow equilibrated with starting buffer(0.4M NaCl, 20 mM sodium phosphate, pH 7.5, 0.02% Tween-20) was added tothe processed milk at a ratio of approximately 1 ml of packed resinbeads per 5-10 mg of lactoferrin in the processed milk. The mixture wasstirred for 20 hours, and the resin beads were isolated bycentrifugation at 1600×g for 5 minutes. Supernatant was removed and thebeads were washed three times with one volume of starting buffer. Theresin was then poured into a column and washed with one volume of 20 mMsodium phosphate, 0.4M NaCl, pH 7.5. Lactoferrin is eluted from thecolumn with a gradient of 0.4-1.0M NaCl in 1.25 (times the column)volume of 20 mM sodium phosphate, pH 7.5, with a flow rate of 10 ml/min.The results of the lactoferrin recovered by this method is shown inTable 4, which tabulates the quality of purified lactoferrin samples asdetermined by gel electrophoresis, spectroscopy, and chromatographicperformance on Mono S™ resin.

                  TABLE 4    ______________________________________    Evaluation of Purified LF preparations                    SDS-PAGE (1)                    Purity/     Spectroscopy (2)                                          Mono S(3)                    Saturation/ Purity/   Purity/    Sample Recovery Cleavage    Saturation                                          Peak III    (Run #)           (%)      (%)         (%)       (%)    ______________________________________    hLF (11)           89       >99/<5/<1   >99/3.1.sup.                                          >99.9/>99    bLF.sup.a (354)           90       >99/<5/<1   >99/8.4.sup.    rhLF.sup.b (24)           80       >99/>90/<1  >99/100   >99.9/>99    ______________________________________     (1) Purity was determined by applying samples of at least 10 μg of     protein to SDSpolyacrylamide (7% w/v) slab gel electrophoresis (SDSPAGE).     Protein bands were visualized by staining with Coomassie Brilliant Blue.     Percentage of iron saturation of lactoferrin was determined by nonreduced     nonboiled SDSPAGE. Cleavage of lactoferrin was determined by reduced     SDSPAGE.     (2) Purity was determined by absorbance spectrum analysis; for     calculations, we used an A 0.1%, 1 cm at 280 nm of 1.1 for unsaturated     (apo) LF, an A 0.1%, 1 cm at 280 nm of 1.4 for saturated (Fe) LF, and an     0.1%, 1 cm at 465 nm of 0.056 for Fe hLF.     (3) Purity was determined by applying 1 mg of protein on a Mono S™ (1     ml) column with a full scale sensitivity of 0.01. Nativity (see below) of     hLF was analyzed by applying 50 μg of protein on the column and     expressed as the % of total hLF that eluted at the peak III position (0.7     NaCl). The elution program was a linear salt (0-1M NaCl) gradient in 30 m     of 20 mM sodium phosphate, pH 7.5.     .sup.a bLF eluted as a single peak with a small shoulder at approximately     0.8M NaCl.     .sup.b rhLF represents transgenic recombinant hLF purified from the milk     of transgenic mice.

Dose-response curves of purified natural hLF and transgenic rhLF asdetermined in a sandwich-type RIA specific for hLF were parallel tostandard curves of commerically available purified human milk-derivedhLF. Amino-terminal protein sequence analysis revealed that the signalsequences of purified natural hLF and transgenic rhLF were correctly andfully removed and showed no N-terminal degradation. Dose-response curvesof purified bLF as determined in a competitive inhibition RIA specificfor bLF were parallel to standard curves of commercially availablebovine milk-derived bLF.

Evaluation of Purified hLF Preparations by Mono S™ Chromatography

During the course of purification studies with transgenic rhLF frommouse milk (linear gradient of 0-1M NaCl in 20 mM sodium phosphate, pH7.5 on a Mono S™ column), we observed that at least approximately 95% oftransgenic rhLF was eluted at 0. 7M NaCl, whereas commercially availablehLF preparations (Sigma and Calbiochem) purified from human milk elutedas three peaks at approximately 0.5, 0.6, and 0.7M NaCl (denoted hLFpeaks I, II, and III, see also Tables 3 and 5). hLF that was purifiedfrom fresh human milk always eluted at approximately 0.7M NaCl. bLFelutes at approximately 0.8M NaCl. Table 5 shows the elution patterns ofvarious lactoferrin and other preparations on a Mono S™ column.

                                      TABLE 5    __________________________________________________________________________    Elution pattern of various LF and other preparations on a Mono S ™    column               Concentrations of NaCl (M) at which    Preparation               a peak is eluted (percentage of    (FPLC Run No.)               bound protein eluting at this position)    __________________________________________________________________________    hLF Sigma 1 (352)                     0.54 (18)                             0.63 (38.7)                                   0.72 (43.6)    hLF Sigma 2 (353)                     0.53 (30.2)                             0.63 (49)                                   0.71 (19.8)    hLF Sigma 3 (1075)       0.61 (5.7)                                   0.69 (94.8)    Calbiochem hLF (116)                     0.54 (18)                             0.62 (17.2)                                   0.72 (63.6)    hLF GPE 1 (1067)               0.68 (100)    2 and 3    hLF GPE 4 (1097) 0.53 (9.1)    0.66 (91.3)    Arg-mod. hLF GPE (30)               no binding    Fe hLF Sigma 1 (396)                     0.52 (2.6)                             0.61 (18.8)                                   0.70 (78)    Fe hLF Sigma 2 (62)      0.60 (4.6)                                   0.68 (95.1)    Fe hLF GPE (824)               0.65 (100)    Deglyc. hLF GPE                 0.7 (>99).sup.    Neura. hLF GPE (894)           0.67 (100)    Tryp. hLF GPE 1 (149)                     0.50 (19.3)                             0.57 (80.3)                                   -- (0)    Tryp. hLF GPE 2 (152)               0.39 (2.3)                     0.48 (49.3)                             0.58 (46.5)                                   -- (0)    Tryp. hLF GPE 3 (165)               0.38 (7.2)                     0.47 (81)                             0.58 (7.3)                                   -- (0)    Trans hLF GPE 1 (1315)   0.63 (5.7)                                   0.69 (94)    Trans hLF GPE 2 (248)          0.66 (>99).sup.    293 rhLF                       0.68 (>99).sup.    293 Unglyc. rhLF               0.69 (>96).sup.    bLF Sigma (351)                0.77 (100)    bLF GPE (421)                  0.76 (100)    Arg-mod. bLF GPE    mLF GPE (1132)               0.26 (100)    mDF GPE (1136)               0.22 (100)    mTF Sigma (548)               no binding    hTF Sigma (57)               no binding    pLFGPE (577)     0.52 (100)    Sheep whey       >0.6 (100)    Goat whey        >0.6 (100)    __________________________________________________________________________

50 μg of protein in 20 mM sodium phosphate, pH 7.5 (buffer A) wasapplied to a Mono S™ column (Pharmacia HR5/5 containing 1 ml of resin)using the FPLC system (Pharmacia). After washing the column with 5 ml ofbuffer A, a linear salt gradient from 0-1.0M NaCl in 30 ml of buffer Awas applied at a flow rate of 1 ml/min. Peaks were monitored byabsorption measurement at 280 nm with a full scale sensitivity of 0.01using a flow cell of 0.2 cm. The following abbreviations are used: hLFSigma 1, purified human milk-derived "native" hLF from Sigma; hLF Sigma2, repeatedly frozen and thawed Sigma 1; HLF Sigma 3, a different lot of"native" hLF from Sigma; Calbiochem hLF, human milk-derived hLF fromCalbiochem; hLF GPE 1, 2, and 3, hLF preparations with iron saturationlevels of about 3% purified by strong cation exchange chromatography offresh human milk samples of three donors with total iron saturation of3%; hLF GPE 4, hLF purified from a human milk sample that had beenstored for one week at 4° C.; FehLF Sigma 1 and 2, different lots ofpurified human milk-derived hLF that is fully saturated with iron bySigma; Arg-mod. hLF GPE, purified human milk-derived hLF that has hadArg residues chemically modified; FehLF GPE, purified human milk-derivedhLF saturated with Fe; Deglyc. hLF GPE, purified human milk-derived hLFthat was completely deglycosylated with N-glycosidase; Neura. hLF GPE,purified human milk-derived hLF that has had sialic acid residuesremoved with neuraminidase; Tryp. hLF GPE 1, 2, and 3, purified humanmilk-derived hLF that was incubated with trypsin (molar ratiohLF:trypsin was 7.5:1) for 1 minute, 3 hours and 24 hours, respectivelyfollowed by addition of soybean trypsin inhibitor; Trans. hLF GPE 1 and2, hLF purified from the milk of mice harboring cDNA (codes for signalsequence of bovine aS1 casein fused to mature hLF) and genomic hLFtransgene constructs expressing rhLF at 0.2 and 2.0 mg/ml, respectively;293 rHLF, recombinant hLF expressed in tissue culture (293 cells); 293Unglyc. rhLF, purified unglycosylated non-cleaved rhLF expressed by 293cells in the presence of tunicamycin; bLF Sigma, purified bovinemilk-derived bLF from Sigma; bLF GPE, bLF purified from fresh bovinemilk by strong cation exchange chromatography; Arg-mod. bLF GPE,purified bLF that has had Arg residues chemically modified; mLF GPE,murine lactoferrin purified from fresh mouse milk by strong cationexchange chromatography; mDF GPE, murine domferrin purified by strongcation exchange chromatography on mouse milk (mDF is an 80 kD proteinthat belongs to the transferrin family); mTF Sigma, purified mouse(sero-)transferrin purchased from Sigma; hTF Sigma, purified humanserotransferrin purchased from Sigma; pLF GPE, porcine lactoferrinpurified from pig milk by strong cation exchange chromatography; Sheepwhey and goat whey, whey fractions prepared from sheep and goat milkrespectively.

In the literature, Makino et al. (1992) J. Chromato. 579: 346 reportsthat three peaks of hLF that elute from a Mono S column at 0.88, 0.97,and 1.05M NaCl represent diferric, monoferric, and apolactoferrin,respectively. However, our results of studies on saturation of hLF withiron indicate that two ferric atoms are coordinately incorporated ineach hLF molecule, so that essentially all hLF from milk is eitherapolactoferrin or diferric. Native hLF (hLF purified from fresh humanmilk; only 3% iron saturated by absorbance measurement) and Fe-hLF(completely saturated with iron as determined by absorbance measurementand non-reduced, non-boiled SDS-PAGE) elute at exactly (within thelimits of experimental resolution) the same position (approximately 0.7MNaCl) from a Mono S™ column at pH 7.5. Completely deglycosylated (withN-glycosidase), neuraminidase-treated, and native hLF elute at exactly(within the limits of experimental resolution) the same position(approximately 0.7M NaCl) from a Mono S™ column at pH 7.5.

The relative amount of hLF peaks I and II increased upon prolonged (4days) dialysis of transgenic mouse whey. In addition, hLF peaks II and Iappeared after limited tryptic proteolysis of native hLF (peak III)before degradation of hLF could be observed by non-reduced or reducedSDS-PAGE. Based on these observations, peaks II and I in hLFpreparations may be generated by limited proteolysis of peak III (nativehLF), such as with a serine protease cleaving at arginine. In line withthis idea were the results of the amino-terminal protein sequenceanalysis of hLF peaks I, II, and III present in commercially availablepurified hLF (see Tables 6 and 7). N-terminal proteolysis of hLF may beimportant as the biological activities of truncated hLF variants may bedifferent from that of native hLF, such as binding of hLF to cellularreceptors, in vivo clearance rate in the circulation, ability to inhibitendocytosis of chylomicron remnants, and/or antibacterial properties.

Tables 6 and 7 show N-terminal sequence analysis of some of the proteinsin Table 5.

                                      TABLE 6    __________________________________________________________________________    N-terminal protein sequence analysis of lactoferrin and some    lactoferrin-related species    (aminoacids 1-25)    __________________________________________________________________________              SEQ ID NO:              1          5          10    __________________________________________________________________________    hLF GPE1  gly--arg--arg--arg--arg--ser--val--gln--trp--xxx--ala--val--ser-              -                                (SEQ ID NO: 1)    hLF Calbi.peakIII                    gly--arg--arg--arg--arg--ser--val--gln--                                               (SEQ ID NO: 2)    hLF Calbi.peakII                       arg--arg--arg--ser--val--gln--trp--xxx--ala--val--ser--              4                                (SEQ ID NO: 3)    hLF Calbi.peakI              arg--arg--ser--val--gln--trp--xxx--ala--val--ser--                                               (SEQ ID NO: 4)    hLF GPE4 peakIII              gly--arg--arg--arg--arg--ser--val--gln--trp--                                               (SEQ ID NO: 5)    Tr.hLF GPE1peakIII              gly--arg--arg--arg--arg--ser--val--gln--trp--xxx--ala--val--ser-              -                                (SEQ ID NO: 6)    bLF cDNA GPE              gly--arg--arg--arg--arg--ser--val--gln--trp--cys--ala--val--ser-              -                                (SEQ ID NO: 7)    Tr.hLF GPE2              gly--arg--arg--arg--arg--ser--val--gln--trp--xxx--ala--val--ser-              -                                (SEQ ID NO: 8)    mLF GPE         lys--ala--thr--thr--val--arg--trp--xxx--ala--val--ser--                                               (SEQ ID NO: 9)    mLF cDNA Teng                    lys--ala--thr--thr--val--arg--trp--cys--ala--val--ser--                                               (SEQ ID NO: 10)    mDF GPE         lys--ala--val--arg--val--gln--trp--xxx--ala--val--ser--                                               (SEQ ID NO: 11)    mTF Sigma    val--pro--asp--lys--thr--val--lys--trp--xxx--ala--val--xxx--                                               (SEQ ID NO: 12)    hTF          val--pro--asp--lys--thr--val--arg--trp--cys--ala--val--ser--                                               (SEQ ID NO: 13)    bLF cDNA Pierce                 ala--pro--arg--lys--asn--val--arg--trp--cys--thr--ile--ser--                                               (SEQ ID NO: 14)    oLF          ala--pro--arg--lys--asn--val--arg--trp--cys--ala--ile--ser--                                               (SEQ ID NO: 15)    pLF          ala--pro--lys--lys--gly--val--arg--trp--cys--val--ile--ser--                                               (SEQ ID NO: 16)    __________________________________________________________________________              SEQ ID NO:              15          20          25    __________________________________________________________________________    hLF GPE1  gln--pro--glu--ala--thr--lys--xxx--phe--gln--trp--gln--arg--                                               (SEQ ID NO: 17)    hLF Calbi.peakI              gln--    Tr.hLF GPE1peakIII              gln--pro--    bLF cDNA GPE              gln--pro--lu--ala--thr--lys--cys--phe--gln--trp--gln--arg--                                               (SEQ ID NO: 18)    Tr.hLF GPE2              gln--pro--glu--ala--xxx--lys--xxx--phe--gln--                                               (SEQ ID NO: 19)    mLF GPE   asn--ser--glu--glu--glu--lys--xxx--leu--arg--trp--gln--                                               (SEQ ID NO: 20)    mLF cDNA Teng              asn--ser--glu--glu--glu--lys--cys--leu--arg--trp--gln--asn--                                               (SEQ ID NO: 21)    mDF GPE   asn--glu--glu--    mTF Sigma glu--his--xxx--asn--ile--lys--   (SEQ ID NO: 22)    hTF       glu--his--glu--ala--thr--lys--cys--gln--ser--phe--arg--asp--                                               (SEQ ID NO: 23)    bLF cDNA Pierce              gln--pro--glu--trp--phe--lys--cys--arg--arg--trp--gln--trp--                                               (SEQ ID NO: 24)    oLF       pro--pro--glu--gly--ser--arg--cys--tyr--gln--trp--gln--lys--                                               (SEQ ID NO: 25)    pLF       thr--ala--glu--tyr--ser--lys--cys--arg--gln--trp--gln--ser--                                               (SEQ ID NO: 26)    __________________________________________________________________________     Proteins given in italics are derived from literature.     Underlined aminoacids represent the basic aminoacid residues.

                                      TABLE 7    __________________________________________________________________________    N-terminal protein sequence analysis of lactoferrin and some    lactoferrin-related species    (aminoacids 26-50)    __________________________________________________________________________            25         30         35    __________________________________________________________________________    hLF cDNA GPE            asn--met--arg--lys--val--arg--gly--pro--pro--val--ser--cys--                                          (SEQ ID NO: 27)    mLF cDNA Teng            glu--met--arg--lys--val--gly--gly--pro--pro--leu--ser--cys--                                          (SEQ ID NO: 28)    hTF     his--met--lys--ser--val--ile--phe--ser--asp--gly--phe--ser--                                          (SEQ ID NO: 29)    bLF cDNA Pierce            arg--met--lys--lys--leu--gly--ala--phe--ser--ile--thr--cys--                                          (SEQ ID NO: 30)    oLF     lys--met--arg--arg--met--     (SEQ ID NO: 31)    pLF     lys--ile--arg--arg--thr--asn--pro--ile--phe--cys--ile--arg--                                          (SEQ ID NO: 32)    __________________________________________________________________________            40         45         50    __________________________________________________________________________    hLF cDNA GPE            ile--lys--arg--asp--ser--phe--ile--gln--cys--ile--gln--ala--ile--                                          (SEQ ID NO: 33)    mLF cDNA Teng            val--lys--lys--ser--ser--thr--arg--gln--cys--ile--gln--ala--ile--                                          (SEQ ID NO: 34)    hTF     val--ala--cys--val--lys--lys--ala--ser--tyr--leu--asp--cys--ile--                                          (SEQ ID NO: 35)    bLF cDNA Pierce            val--arg--arg--ala--phe--ala--leu--glu--cys--ile--arg--ala--ile--                                          (SEQ ID NO: 36)    pLF     arg--ala--ser--pro--thr--asp--cys--ile--arg--ala--ile--                                          (SEQ ID NO: 37)    __________________________________________________________________________

On the basis of the elution patterns of Table 5, the N-terminal proteinsequence data of Tables 6 and 7 as well as those of transferring andlactoferrins published in the literature, we conclude that theamino-terminal sequences of transferrins/lactoferrins determine thebinding characteristics of these molecules to strong cation exchangeresins and that native (peak III) hLF can be purified from degradationproducts (peaks I and II) by strong cation exchange chromatography.Thus, strong cation exchange chromatography can be used for qualitycontrol assessment of hLF by separating and quantitatively detecting theamounts of native hLF and degradation products (peaks I and IImaterial), and for preparative purification of native hLF from suchdegradation products, such as for pharmaceutical formulation ofhomogeneous native hLF or purified peak I or peak II material, ifdesired. On the basis of the differential elution patterns of nonhumanand human lactoferrin species as presented in Table 5 as well as fromexperiments with mixtures of purified hLF and bLF, and hLF-spiked bovinemilk on Mono S™ (see below), we also conclude that separation oftransgenically produced rhLF from nonhuman lactoferrin (e.g., bLF) canbe accomplished with strong cation-exchange chromatography by the use ofa salt gradient or stepwise elution with buffers of increasing ionicstrength (salt concentration). It is to be noted that transgenic rhLFproduced in the milk of transgenic mice eluted at the same ionicstrength as hLF derived from human milk. Furthermore, all proteins fromsheep and goat whey fractions that had bound to Mono S™ were eluted anNaCl concentrations lower than 0.6M NaCl (Table 5). This indicates thattransgenic rhLF can be resolved from sheep and goat lactoferrin speciesby strong cation-exchange chromatography.

Chromatographic Separation of hLF and bLF

Table 8 shows the elution patterns of hLF and bLF on Mono S™ with linearsalt gradients demonstrating the different salt strengths at which hLFand bLF elute.

                  TABLE 8    ______________________________________    Effect of the pH on the elution pattern of HLF and BLF on Mono S ™                                          Difference    FPLC                    hLF    bLF    bLF-hLF    run no.  Buffer         (M NaCl)      (M NaCl)    ______________________________________    943      20 mM Na acet pH 5.0                            0.72    944           "                0.78    945           "         0.72   0.78   0.06    955      20 mM Na phos pH 6.0                            0.69    956           "                0.76    957           "         0.69   0.76   0.07    934      20 mM Na phos pH 7.5                            0.66    938           "                0.73    940           "         0.66   0.73   0.07    948      20 mM eth am pH 9.5                            0.57    949           "                0.63    950           "         0.57   0.63   0.06    ______________________________________     Fifty μg of protein (HLF Sigma 3 and/or BLF Sigma) in buffers of     different pH (buffer A) was applied to the column (Pharmacia HR5/5     containing 1 ml of equilibrated resin beads) using the FPLC system of     Pharmacia. After washing the column with 5 ml of buffer A, a linear salt     gradient from 0-1.0M NaCl in 30 ml of buffer A was applied at a flow rate     of 1 ml/minute. Peaks were monitored by absorption measurement at 280 nm.

Since the difference in elution patterns was relatively pH insensitiveat the pH values tested, the optimization of the separation of hLF andbLF on Mono S™ was determined at near physiological pH (pH7.5 ). Asshown in FIG. 1, 100 μg of HLF Sigma 3 (panel A) or ELF Sigma (panel B)in buffer A (20 mM sodium phosphate, pH 7.5 ) was applied to the Mono S™column (Pharmacia HR5/5 containing 1 ml of equilibrated resin beads)using the FPLC system of Pharmacia. After washing the column with 5 mlof buffer A, a linear salt gradient from 0-1.0M NaCl in 30 ml of bufferA was applied at a flow rate of 1 ml/minute. Peaks were monitored byabsorption measurement at 280 nm (full scale 0.02).

FIG. 2 shows the differential elution pattern of hLF and bLF on Mono Smwith a stepwise elution program (sequential increases of saltconcentration to 0.6 and 1.0M NaCl). 100 μg of hLF Sigma 3 (panel A) orbLF Sigma (panel B) in buffer A was applied to the Mono S™ column(Pharmacia HR5/5 containing 1 ml of equilibrated resin beads) using theFPLC system of Pharmacia. After washing the column with 5 ml of bufferA, the salt concentration was stepwise increased from 0M to 0.6M NaCl inbuffer A, pH 7.5 at a flow rate of 1 ml/minute. After 10 minutes,another stepwise increase from 0.6M to 1.0M NaCl in buffer A was appliedat 1 ml/minute. Peaks were monitored by absorption at 280 nm.

FIG. 3 shows the substantial purification of hLF and bLF by resolutionof a mixture of purified proteins on a Mono S™ column either with alinear salt gradient or stepwise elution. 100 μg of hLF and 100 μg ofbLF were each applied to the column in buffer A and eluted by linearNaCl gradient (panel A) or stepwise elution (panel B) as describedabove. The major hLF peak elutes at 0.67M NaCl and the major bLF peakelutes at 0.75M NaCl under the conditions used for linear gradientelution (FIG. 3, panel A). With the stepwise elution program, the majorhLF peak elutes in 0.6M NaCl step buffer and the major bLF peak elutesin the 1.0M NaCl step buffer (FIG. 3, panel B).

FIG. 4 shows specific radioimmunoassays to quantitate hLF and bLF inelution fractions of the gradient-wise (panel A) and stepwise (panel B)elutions under FIG. 3. The purity of top fractions of hLF and bLFexceeds approximately 95% as determined by RIA (i.e., less than about 5%cross contamination). Resolution of hLF from bLF was somewhat betterwith stepwise elution than linear gradient elution.

Purified hLF was added to raw bovine milk and used to determine thechromatographic purification of hLF from bLF in bovine milk by using astrong cation exchange resin. Bovine milk to which sodium phosphate, pH7.5 (20 mM), NaCl (0.4M), Tween-20 (0.02%) and either hLF (100 μg/ml) orbuffer alone had been added was stirred for 20 minutes at roomtemperature (final pH was 6.6). Skimmed milk (obtained by centrifugationat 15,000×g for 30 minutes at 4° C.) was adjusted to pH 4.7 with 1N HCland incubated at 40° C. for 30 minutes. Whey fraction (obtained bycentrifugation at 15,000×g for 30 minutes at 4° C.) was adjusted to pH7.5 with 1N NaOH, and further clarified by centrifugation at 15,000×gfor 5 minutes at 20° C. followed by filtration through a 0.22 μm filter.One ml samples of whey were applied to the Mono S™ column equilibratedwith 0.4M NaCl, 20 mM sodium phosphate, pH 7.5. The column was thenwashed with 18 ml of 0.4M NaCl, 20 mM sodium phosphate, pH 7.5 at 1ml/min. Peaks were monitored by absorption measurement at 280 nm (fullscale 0.01). FIG. 5 shows the chromatograms when a linear salt gradientfrom 0.4-1.0M NaCl in 18 ml of 20 mM sodium phosphate, pH 7.5 wassubsequently applied; panel A shows bovine whey with bLF only (unspiked)and panel B shows bovine whey containing bLF and hLF (spiked). FIG. 6shows the chromatograms when the salt concentration was stepwiseincreased from 0.4M to 0.6M NaCl in 20 mM sodium phosphate, pH 7.5;after 10 minutes, another stepwise increase from 0.6M to 1.0M NaCl in 20mM sodium phosphate, pH 7.5 was applied; panel A shows bovine whey withbLF only (unspiked) and panel B shows bovine whey containing bLF and hLF(spiked). The resolution of hLF from bLF with stepwise elution wasbetter than that seen with a linear salt gradient under the conditionstested.

The volume as well as salt strength of an elution buffer required tostart and establish complete elution of hLF relate to the amount of hLFbound to the column. A small increase (e.g., stepwise from 0.4M to 0.5MNaCl) in salt concentration will readily and preferentially startelution of hLF when the column is loaded with increasing amounts of hLF.It was observed that the more hLF bound to the resin, the lower the saltconcentration required to start elution of hLF and the greater thetailing of the hLF peak that occurred.

Effects of Salt Concentration on Elution Volume

100 μg of hLF were loaded on a Mono S™ column and elution buffers ofvarying NaCl concentration in 20 mM sodium phosphate, pH 7.5 wereapplied to completely elute the bound hLF. Table 9 shows the volumes (inml) of each of the salt concentrations required for complete elution ofthe hLF.

                  TABLE 9    ______________________________________    Elution Volume Dependence on Salt Concentration    NaCl    (M)     Volume (ml) required for complete elution    ______________________________________    0.4     165    0.5     17    0.6     4.9    0.7     2.5    0.8     1.7    0.9     1.5    ______________________________________

With lower salt concentrations, the sharpness of the peaks eluted wasnoted to decrease. These results indicate that the volume of washingbuffer (e.g., 0.4M NaCl) during large scale purification should belimited, since washing the resin with large volumes relative to thevolume of the packed resin under the tested conditions would completelyelute the bound hLF.

Scale-up Purification of hLF

Varying amounts of hLF were loaded onto a 1 ml Mono S™ column and alinear salt gradient (0-1.0M NaCl in 30 ml of 20 mM sodium phosphate, pH7.5) was applied at 1 ml/min. The NaCl concentration at which elution ofhLF was observed to begin as well as that at which hLF elution wascomplete was recorded. Table 10 shows the results.

                  TABLE 10    ______________________________________    Elution patterns of varying amounts    of HLF bound to a Mono S ™ column    Amount of             NaCl concentration (M)    hLF bound             at which elution    to Mono S ™             starts/appears complete                              Peak shape    ______________________________________       50 μg              0.69            very sharp (A = 0.01)    (run 1144)                95% protein in 1.5 ml     8,500 μg             0.54/0.73        broad (A = 0.5)    (run 1145)                95% protein in 4.5 ml    20,000 μg             0.44/0.7         broader (A = 1.0)    (run 1152)                95% protein in 7.2 ml    36,500 μg             0.29/0.73        very broad (A = 2.0)    (run 1146)                95% protein in 11.0 ml    ______________________________________

FIG. 7 depicts the relationship between the amount of hLF bound to a 1ml Mono S™ column and the NaCl concentration at which elution of hLF wasobserved to begin when a linear salt gradient (0-1.0M NaCl in 30 ml of20 mM sodium phosphate, pH 7.5) was applied at 1 ml/min. The data ofFIG. 7 give an indication of the maximum binding capacity (mg hLF boundper ml of resin) of Mono S™. Experiments with S Sepharose™ Fast Flowproduced similar results.

Affinity Chromatography on Concanavalin A

Glycosylation differences which exist between hLF and bLF can be used toseparate hLF to bLF by lectin chromatography. hLF bound to Con A couldbe completely eluted with 50 mM α-methyl-D mannopyranozide in 50 mM TrisHCl, pH 8.0, 0.14M NaCl, whereas bLF was not significantly eluted, evenwhen 200 mM α-methyl-D-mannopyranozide was used. Con A chromatographycan thus be used to separate hLF from bLF, such as removing traces ofbLF in hLF preparations made by strong cation exchange chromatographyi.e., an alternative to rechromatography on a strong cation exchangeresin. Examples of strong cation exchange resins include, but are notlimited to:

    ______________________________________    RESIN                 SUPPLIER    ______________________________________    S Sepharose Fast Flow Pharmacia    SP Sephadex C-50      Pharmacia    SP Sephadex C-25      Pharmacia    Mono S ™           Pharmacia    SP Sepharose Fast Flow                          Pharmacia    SP Sepharose Big Beads                          Pharmacia    A9 50 W X2            BioRad    A9 50 W X4            BioRad    A9 50 W X8            BioRad    A9 50 W X12           BioRad    A9 50 W X16           BioRad    Protein Pak SP 15 HR  Millipore/Waters    Protein Pak SP 40 HR  Millipore/Waters    Parcosil PepKat       Serva    Parcomer PekKat       Serva    Fractogel EMD SO.sub.3 650 (M)                          Merck    ______________________________________

Separation of hLF and bLF with HIC

Hydrophobic interaction chromatography (HIC) separates proteins on thebasis of differences in hydrophobic surfaces on the molecules. Thematrix contains a hydrophobic ligand (like a phenyl or butyl group). Inthe presence of high salt concentrations (e.g. >1M (NH₄)₂ SO₄),hydrophobic surfaces on the molecules are exposed and will bind to theresin. Proteins are eluted by decreasing the salt concentration.

It has been reported by Yoshida (J. Diary Sci. 72 (1989) 1446-1450) thatbLF can bind to a butyl Toyopearl 650M column. bLF does not elute usingdeionized water, but using 0.25M acetic acid. LF from human tears wasalso reported to bind to HIC columns (Baier et al (1990) J. Chromat.525, 319-328). Using reverse-phase (phenyl) HPLC, Hutchens et al (PNASUSA 88 (1991) 2994-2998) showed that human LF and LF degradationproducts can be separated. Tryptic fragments of human LF could beseparated with reverse-phase (octadecyl) HPLC (Shimazaki et al (1993) J.Diary Sci. 76, 946-955). The prior art does not suggest that hLF and bLFcan be separated with HIC. The invention accordingly includes the use ofHIC in separating hLF and bLF and corresponding methods.

50 μg pure hLF or bLF, diluted in buffer A (50 mM NaPi pH 7.5, 2.5M(NH₄)₂ SO₄), are loaded on a 1 ml Phenyl Sepharose FF (high sub;Pharmacia) column at a flow rate of 0.2 ml/min. After 5 ml wash withbuffer A, all hLF is eluted blockwise with about 11 ml of 80% buffer(buffer B=50 mM NaPi pH 7.5. No elution is observed of bLF under theseconditions. All bFL elutes with a subsequent block (with 5 ml) of 100%buffer B. This result clearly shows that hLF and bLF can be separatedwith this technique. A disadvantage is the relatively high saltconcentrations in which the proteins elute. The technique can be used tofurther separate transgenic hLF from bLF obtained after S Sepharosechromatography.

Mouse Domferrin

Another aspect of the invention is an isolated mouse domferrin proteinhaving the N-terminal sequencelys-ala-val-arg-val-gln-trp-xxx-ala-val-ser-asn-glu-glu, (SEQ ID NO:11)a very approximate molecular weight by SDS PAGE of 80 kD, and an elutionprofile from MonoS whereby the protein is obtained at a concentration ofabout 0.22M salt.

The invention includes preparation of such proteins by cation exchangechromatography.

Some preferred properties of mouse domferrin can be summarized:

1. Elutes from Mono S at a unique position (i.e. 0.22M salt; mouselactoferrin elutes at 0.26M salt).

2. The protein has a unique N-terminal sequence, different from mouselactoferrin, and different from mouse transferrin (and other lactoferrinand transferrin species)--see tables above.

3. The migration pattern on SDS-PAGE indicates and ˜80 kD protein.(Fuzzy bands are observed, probably due to glycosylation differences.)

Additional information:

4. In immunodiffusion assays no crossreactivity of mouse domferrin withanti-mouse transferrin or anti human lactoferrin antibodies is observed.

5. Mouse domferrin has no peroxidase activity (tested in alactoperoxidase assay).

Although the present invention has been described in some detail by wayof illustration for purposes of clarity of understanding, it will beapparent that certain changes and modifications may be practiced withinthe scope of the claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 17    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..25    (D) OTHER INFORMATION: /note= "hLF GPE1 N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GlyArgArgArgArgSerValGlnTrpXaaAlaValSerGlnProGlu    151015    AlaThrLysXaaPheGlnTrpGlnArg    2025    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..8    (D) OTHER INFORMATION: /note= "hLF Calbi.peakIII N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GlyArgArgArgArgSerValGln    15    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..11    (D) OTHER INFORMATION: /note= "hLF Calbi.peakII N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ArgArgArgSerValGlnTrpXaaAlaValSer    1510    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..11    (D) OTHER INFORMATION: /note= "hLF Calbi.peakI N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ArgArgSerValGlnTrpXaaAlaValSerGln    1510    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..9    (D) OTHER INFORMATION: /note= "hLF GPE4 peakIII N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GlyArgArgArgArgSerValGlnTrp    15    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..15    (D) OTHER INFORMATION: /note= "Tr.hLF GPE1peakIII N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GlyArgArgArgArgSerValGlnTrpXaaAlaValSerGlnPro    151015    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 50 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..50    (D) OTHER INFORMATION: /note= "hLF cDNA GPE N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GlyArgArgArgArgSerValGlnTrpCysAlaValSerGlnProGlu    151015    AlaThrLysCysPheGlnTrpGlnArgAsnMetArgLysValArgGly    202530    ProProValSerCysIleLysArgAspSerPheIleGlnCysIleGln    354045    AlaIle    50    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /note= "Tr.hLF GPE2 N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GlyArgArgArgArgSerValGlnTrpXaaAlaValSerGlnProGlu    151015    AlaXaaLysXaaPheGln    20    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..22    (D) OTHER INFORMATION: /note= "mLF GPE N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    LysAlaThrThrValArgTrpXaaAlaValSerAsnSerGluGluGlu    151015    LysXaaLeuArgTrpGln    20    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..48    (D) OTHER INFORMATION: /note= "mLF cDNA Teng N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    LysAlaThrThrValArgTrpCysAlaValSerAsnSerGluGluGlu    151015    LysCysLeuArgTrpGlnAsnGluMetArgLysValGlyGlyProPro    202530    LeuSerCysValLysLysSerSerThrArgGlnCysIleGlnAlaIle    354045    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..14    (D) OTHER INFORMATION: /note= "mDF GPE N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    LysAlaValArgValGlnTrpXaaAlaValSerAsnGluGlu    1510    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..18    (D) OTHER INFORMATION: /note= "mTF Sigma N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    ValProAspLysThrValLysTrpXaaAlaValXaaGluHisXaaAsn    151015    IleLys    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 49 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..49    (D) OTHER INFORMATION: /note= "hTF N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    ValProAspLysThrValArgTrpCysAlaValSerGluHisGluAla    151015    ThrLysCysGlnSerPheArgAspHisMetLysSerValIlePheSer    202530    AspGlyPheSerValAlaCysValLysLysAlaSerTyrLeuAspCys    354045    Ile    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 49 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..49    (D) OTHER INFORMATION: /note= "bLF cDNA Pierce N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    AlaProArgLysAsnValArgTrpCysThrIleSerGlnProGluTrp    151015    PheLysCysArgArgTrpGlnTrpArgMetLysLysLeuGlyAlaPhe    202530    SerIleThrCysValArgArgAlaPheAlaLeuGluCysIleArgAla    354045    Ile    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..29    (D) OTHER INFORMATION: /note= "oLF N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    AlaProArgLysAsnValArgTrpCysAlaIleSerProProGluGly    151015    SerArgCysTyrGlnTrpGlnLysLysMetArgArgMet    2025    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..47    (D) OTHER INFORMATION: /note= "pLF N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    AlaProLysLysGlyValArgTrpCysValIleSerThrAlaGluTyr    151015    SerLysCysArgGlnTrpGlnSerLysIleArgArgThrAsnProIle    202530    PheCysIleArgArgAlaSerProThrAspCysIleArgAlaIle    354045    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..14    (D) OTHER INFORMATION: /note= "domferrin N-terminus"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    LysAlaValArgValGlnTrpXaaAlaValSerAsnGluGlu    1510    __________________________________________________________________________

We claim:
 1. A method for the separation of human and bovine lactoferrinwhich comprises subjecting a mixture including the said lactoferrins tohydrophobic interaction chromatography, and eluting human lactoferrinseparately from bovine lactoferrin.
 2. The method of claim 1, whereinthe mixture is milk or a milk fraction from a transgenic bovineexpressing human lactoferrin in its milk.
 3. The method of claim 1,wherein said hydrophobic interaction chromatography is carried out usinga resin containing a ligand that comprises a phenyl group or a butylgroup.
 4. The method of claim 3, wherein the ligand comprises a phenylgroup and said ligand is linked to an agarose support.
 5. The method ofclaim 2, wherein the human lactoferrin is eluted in 40 mM NaPO₄ buffer.6. A method for substantially purifying human lactoferrin from a mixturecontaining both human lactoferrin and bovine lactoferrin, said methodcomprising the steps of:contacting the mixture containing humanlactoferrin and bovine lactoferrin with a hydrophobic chromatographyresin under elevated ionic strength conditions, whereupon the humanlactoferrin associates with said resin to form human lactoferrin-resincomplexes and the bovine lactoferrin associates with said resin to formbovine lactoferrin-resin complexes; and, eluting the human lactoferrinfrom the human lactoferrin-resin complexes under conditions in which thebovine lactoferrin is not eluted from the bovine lactoferrin-resincomplexes, whereupon human lactoferrin substantially free from bovinelactoferrin is obtained.
 7. The method of claim 6, wherein the mixtureis milk or a milk fraction from a transgenic bovine expressing humanlactoferrin in its milk.
 8. The method of claim 6, wherein thehydrophobic chromatography resin contains a ligand that comprises aphenyl group or a butyl group.
 9. The method of claim 8, wherein theligand comprises a phenyl group and said ligand is linked to an agarosesupport.
 10. The method of claim 8, wherein the human lactoferrin iseluted in 40 mM NaPO₄ buffer.
 11. The method of claim 10, wherein themixture comprises human lactoferrin and bovine lactoferrin and isadjusted to an ionic strength greater than that of 1M NH₄ SO₄.